Ammopiptanthus mongolicus cold induced protein as well as preparation method and application thereof

Abstract

The invention discloses an ammopiptanthus mongolicus cold induced protein as well as a preparation method and application thereof, and belongs to the technical field of enzyme activity protection. The amino acid sequence of the ammopiptanthus mongolicus cold-induced protein is as shown in SEQ ID No.1, and the sequence of the coding gene of the ammopiptanthus mongolicus cold-induced protein is as shown in SEQ ID No.2. And introducing the coding gene of the ammopiptanthus mongolicus into recipient cells, and carrying out protein expression and purification to obtain the ammopiptanthus mongolicus cold-induced protein. The ammopiptanthus mongolicus cold-induced protein can protect the enzyme activity of lactic dehydrogenase under the condition of copper ions, the protein is a phytogenic enzyme activity protective agent and is safer and wider in application field compared with an organic enzyme activity protective agent, and the commercialized application of the ammopiptanthus mongolicus cold-induced protein enables enzymes used in the industries of food, medical treatment, chemical engineering and the like to be more efficient and products to be safer.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme activity protection, and particularly relates to a cold-induced protein of Ammopiptanthus mongolicus and a preparation method and application thereof. Background Art

[0002] Mongolica mongolica Ammopiptanthus mongolicus ) is a plant of the genus Ammopiptanthus in the Leguminosae family. It is a relict plant in the Tertiary desert area and a relict plant of the Quaternary glacier. In China, most of it is found in the arid areas of Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Gansu Province, that is, the geographical location is 36°27′~42°01′ N, 102°36′~108°49′ E. It is a rare and endangered plant under national second-level key protection. The climate characteristics of this area are mainly arid continental climate, with long sunshine hours and sufficient sunshine, but it is far away from the ocean, resulting in very little precipitation and arid climate. Due to the extreme temperature difference, the annual evaporation is 2246.3 mm, which aggravates the drought in the area. At the same time, the soil in the area is mainly sandy gravel and gravel soil, with a shallow soil layer, a large number of pebbles and gravels, and poor water control ability, which makes the soil layer barren and severely eroded, seriously affecting the growth of vegetation in the area. Plants lose water in drought, saline-alkali and other environments, which leads to increased intracellular salt and heavy metal ion content, which in turn leads to denaturation, reduced vitality or inactivation of proteins and enzymes, and ultimately hinders plant growth and development or even plant death. The fact that Ammopiptanthus mongolicus can survive in such a harsh environment suggests that it may have a unique coping strategy.

[0003] Enzymes are widely used in people's production and life. For example, bromelain is used in food processing to change the sensory perception of food; amylase and cellulase are added to feed to improve nutritional value; enzymes are added to washing powder to improve washing efficiency, etc. The essence of enzymes is usually protein. Changes in fixed spatial structure lead to reduced or inactivated enzyme activity. The level of enzyme activity seriously affects production efficiency and the speed of life activities. Enzymes are usually added with enzyme activity protectants during production and life to maintain the stability and function of the enzymes. Commonly used enzyme protectants include a series of organic solvents, such as dimethylsulfoxide (DMSO), dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), polyoxyethylene sorbitan fatty acid esters 20, Tween20, etc. These molecules are toxic to varying degrees and need to be used with caution in people's production or life. This requires safe and non-toxic enzyme activity protectants to be applied in different fields. Therefore, the development and use of safe and non-toxic plant-derived enzyme activity protectants will provide more opportunities for the development of related fields. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a cold-induced protein of Ammopiptanthus mongolicus and a preparation method and application thereof, which solves the problem that the existing enzyme activity protectants are organic reagents, which have different degrees of toxicity and limit the application field of enzymes.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a cold-induced protein of Ammopiptanthus mongolicus, the amino acid sequence of the cold-induced protein of Ammopiptanthus mongolicus is shown in SEQ ID No.1.

[0006] Based on the above technical solution, the present invention can also be improved as follows.

[0007] Furthermore, the coding gene sequence of the cold-induced protein of Ammopiptanthus mongolicus is shown in SEQ ID No.2.

[0008] Furthermore, the method for preparing the cold-induced protein of Ammopiptanthus mongolicus is to introduce the coding gene shown in SEQ ID No. 2 into the receptor cell, express and purify the protein, and obtain the product.

[0009] Furthermore, the cold-induced protein of Ammopiptanthus mongolicus was expressed using a prokaryotic expression system.

[0010] Further, (1) obtaining the target gene: using the cDNA obtained from Ammopiptanthus mongolicus as a template, using primers AmCIP-F and AmCIP-R to perform PCR amplification, and recovering the PCR product to obtain the target gene; the sequences of primers AmCIP-F and AmCIP-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; (2) Double digestion and ligation: Double digestion of the pET28a vector and the target gene obtained in step (1) with restriction endonucleases, respectively, to obtain the double digestion vector and the double digestion target gene; and ligation of the double digestion vector and the double digestion target gene with T4 DNA ligase; (3) Obtaining recombinant plasmid: The ligation product obtained in step (2) was transformed into Escherichia coli DH5α for resistance screening, followed by double enzyme digestion and sequencing identification, and the plasmid was extracted to obtain the recombinant plasmid pET28a- AmCIP ; (4) Expression and purification of target protein: Recombinant plasmid pET28a- AmCIP The protein was transformed into Escherichia coli BL21 and expanded, and IPTG was used to induce the expression of the cold-induced protein of Ammopiptanthus mongolicus; the protein was purified using the HIS tag on the protein.

[0011] Furthermore, the application of cold-induced protein of Ammopiptanthus mongolicus in enzyme activity protection.

[0012] Furthermore, the cold-induced protein of Ammopiptanthus mongolicus protected the enzymatic activity of lactate dehydrogenase.

[0013] Furthermore, the cold-induced protein of Ammopiptanthus mongolicus protected the enzymatic activity of lactate dehydrogenase under heavy metal conditions.

[0014] Furthermore, the cold-induced protein of Ammopiptanthus mongolicus protected the enzymatic activity of lactate dehydrogenase under copper ion conditions.

[0015] Furthermore, the effective concentration of cold-induced protein of A. mongolica was 3~5 µmol / L.

[0016] The beneficial effects of the present invention are as follows: enzymes are prone to reduced activity or inactivation, and enzyme activity protectants are usually added during use or storage. Commonly used enzyme activity protectants are organic reagents, which have varying degrees of toxicity, limiting the application areas of enzymes. The development and use of safe and non-toxic plant-derived enzyme activity protectants will provide more opportunities for the development of related fields. Mongolian sand boxwood cold-induced protein can protect the activity of lactate dehydrogenase under copper ion conditions. This protein is a plant-derived enzyme activity protectant that is safer than organic enzyme activity protectants and has a wider range of applications. Its commercial application will make enzymes used in food, medical, chemical and other industries more efficient and the products safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 for AmCIP Detection of gene amplification products; Figure 2 For the prokaryotic expression vector pET28a- AmCIP Spectrum of Figure 3 The AmCIP protein was expressed and purified in prokaryotes; Figure 4 AmCIP protein protects lactate dehydrogenase; Figure 5 The AmCIP protein protects lactate dehydrogenase under heavy metal copper ion stress. DETAILED DESCRIPTION

[0018] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0019] Example 1 The specific steps for raising seedlings of Ammopiptanthus mongolicus are as follows: (1) Soak the seeds of Ammopiptanthus mongolicus in 75% alcohol by volume for 10 min, stirring appropriately; then wash them with an appropriate amount of distilled water for 6 times, each time for 1 min; then soak the seeds in warm water at 30°C for 1 day, changing the water twice during this period; (2) Place the sterilized seeds in groups of 10 evenly in a culture dish using tweezers and add water to a depth of 2 cm. Cultivate the seeds in a culture room at 25°C with a light intensity of 8 h for about 1 week until they germinate and grow radicles. (3) Transfer the germinated Ammopiptanthus mongolicus seeds to about 2 cm below the culture soil and culture them in a culture room at 25°C with a light intensity of 12 h. Water them once a day to ensure soil moisture and maintain normal seed growth. Continue to culture and grow them until they reach the four-leaf stage.

[0020] Example 2 Cold-induced protein from Ammopiptanthus mongolicus AmCIP The specific steps for gene cloning and sequence determination are as follows: (1) The leaves of Ammopiptanthus mongolicus cultured in Example 1 were quickly frozen with liquid nitrogen and ground into powder, about 100 mg were taken and placed in a 1.5 mL centrifuge tube, and the total RNA of Ammopiptanthus mongolicus was extracted using the Trizol method; (2) Use PrimeScriptTM RT regeant Kit (TaKaRa, Beijing) to remove genomic DNA and reverse transcribe RNA into cDNA; (3) Use Primer 5 software to AmCIPThe CDS sequence of the gene was used as a template to design primers; the upstream primer sequence AmCIP-F: 5'-ATGGCAGGTATCATCAACAAGATTGGG-3' (SEQ ID No. 3), the downstream primer sequence AmCIP-R: 5'-CTAGTCACTGTCACTGCTGCTGCTGTC-3' (SEQ ID No. 4); (4) Take out various amplification reagents from the -20°C refrigerator and place them on ice to melt. Prepare the PCR system according to the reagents and amounts shown in Table 1; Table 1 PCR system

[0021] (5) Place the prepared PCR system into a PCR instrument, set the parameters according to the PCR reaction program shown in Table 2, and perform PCR.

[0022] Table 2 PCR program

[0023] (6) After the PCR is completed, add 2.5 μL of 10× Loading Buffer to the reaction system and use 1.25% agarose for detection. The results are as follows: Figure 1 As shown in the figure (lanes 1 and 2 in the figure represent the PCR reaction products of two tubes); the single band sequence was carefully cut with a scalpel and sent to Shenggong Bioengineering Co., Ltd. for sequencing, and the cold-induced protein of Ammopiptanthus mongolicus was detected. AmCIP The gene sequence is as follows: 5'-ATGGCAGGTATCATCAACAAGATTGGGGAGACCCTTCATGTGGGAGGGAACAAGAAAGAGGATGAGCACAAGGTGGAGAGCCATGGTGATTACAAGGGTGAGCGCCATGATGAAAAAACAGGGGTGCTTCATGGGCTTGGAGGGCACAAGGGAGAGAGCCATGGTGATTACAAGGGTGAAAACACGGGGGTGCTTTCATGGGCTTGGAGGGCACAAGGGAGAGAGCCGTGGTGAT TACAAGGGTGAAAACACGGGGGTGCTTCATGGGCTTGGAGGGCACAAGCCAGATCATTATGGACATGGTGAAGAGCACAAGGAAGGCTTTGTAGACAAGATCAAGGACAAGATCCATGGTGACCCTGATCATGTCAAGGGCGAGGGCGTCGTCAAGAAGAAGAAGGACAAGAAGAAGCATGAACATGGCCATGAGCATGGACATGACAGCAGCAGCAGTGACAGTGACTAG-3' (SEQ ID No.2).

[0024] Example 3 The specific steps for constructing the AmCIP prokaryotic expression vector are as follows: Obtained from sequencing AmCIP The CDS sequence of the gene was combined with the restriction sites on the pET28a vector, and the restriction sites and upstream and downstream primers of the gene were analyzed by Primer Primer 5.0 software. AmCIP The two restriction sites added at both ends of the CDS sequence of the gene are Where? Ⅰ and Hin dⅢ, and add a protective base to the 5' end of the primer. The underline is the restriction site. The primer design is as follows: 28a-CIP-F( Where? Ⅰ): 5'-GGAATTC CATATG ATGGCAGGTATCATCAACAAGATTGGG-3' (SEQ ID No. 5); 28a-CIP-R( Hin dⅢ): 5'-CCC AAGCTT CTAGTCACTGTCACTGCTGCTGCTGTC-3' (SEQ ID No. 6).

[0025] (1) Amplification of target gene TaKaRa's Primer STAR HS high-fidelity enzyme was used for amplification. After PCR amplification, 1.25% agarose gel electrophoresis was performed. The gel block containing the target gene was cut out with a scalpel under ultraviolet light, and the target gene was purified and recovered using a universal DNA purification and recovery kit (TIANGEN, Beijing).

[0026] (2) Enzyme digestion of target gene and vector Use restriction enzymes Where? Ⅰ and Hin The PCR products and pET28a empty plasmids were digested by dⅢ at 37℃ overnight. The double digestion system is shown in Table 3. After digestion, the DNA was purified and recovered according to the instructions of Tiangen Universal DNA Purification and Recovery Kit (TIANGEN, Beijing).

[0027] Table 3 Double enzyme digestion system

[0028] (3) Connection and transformation of vector and target gene TaKaRa's T4 ligase was used to connect the purified target gene recovered after enzyme digestion and the linearized vector, and the connection system is shown in Table 4. The above system was mixed, centrifuged instantly, and placed in a 16°C constant temperature water bath for connection overnight.

[0029] Table 4 Connection system

[0030] 10 µL of the ligation product was transformed into E. coli In the DH5α competent state, spread on an LB plate containing 50 µg / mL Kanmycin, invert and incubate at 37°C for about 12 hours. Pick a single clone in a 1.5 mL centrifuge tube, add 1 mL of LB liquid culture medium containing 50 µg / mL Kanmycin, and incubate at 37°C in a constant temperature shaker for 5 hours.

[0031] (4) PCR detection of positive single clones in bacterial liquid Tiangen Taq enzyme was used for bacterial liquid PCR detection, and 1% agarose gel electrophoresis was performed to select the single clone identified as positive by PCR. After expansion culture, the plasmid DNA was extracted using Tiangen plasmid extraction kit. It was sent to Shanghai Shenggong Company for sequencing, and restriction endonucleases were used at the same time. Where? Ⅰ and Hin The plasmid was digested by dⅢ at 37℃ overnight to verify the correctness of the vector construction. The constructed plasmid pET28a- AmCIP The schematic diagram is as follows Figure 2 shown.

[0032] (5) Heat shock transformation of plasmid into E. coli BL21 and positive detection Heat shock transformation was used to transform 1 µL of plasmid into the prokaryotic expression strain. E. coli In the competent state of BL21, spread on LB plates containing 50 µg / mL Kanmycin, invert and incubate at 37℃ for about 12 hours. Pick a single clone into a 1.5 mL centrifuge tube, add 1 mL of LB liquid culture medium containing 50 µg / mL Kanmycin, and incubate at 37℃ for 5 hours. Use primers 28a-CIP-F and 28a-CIP-R to perform positive detection on the expanded single clone.

[0033] Example 4 The specific steps for prokaryotic expression and purification of AmCIP protein are as follows: (1) 1 mL of overnight culture containing pET28- AmCIP Plasmid E. coli BL21 bacterial solution was inoculated into 100 mL LB medium containing 50 µg / mL Kanmycin at a ratio of 1:100, and cultured in a 37°C shaker at 200 r / min; when the OD value of the bacterial solution reached about 0.6, IPTG with a final concentration of 0.5 mmol / L was added, and the culture was induced at 37°C for 2 h in a shaker at 200 r / min; the cells were collected by centrifugation at 12000 r / min for 10 min, the supernatant was discarded, 50 mL PBS buffer was added to wash the cells, and the cells were collected by centrifugation at 12000 r / min for 10 min.

[0034] (2) Add 10 mL of PBS buffer to the bacterial pellet to resuspend the cells, and add phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, at a final concentration of 1 mmol / L. Ultrasonicate the cells in an ice bath at a power of 400 W for 20 min (9 s of ultrasonication and 5 s of pause as one cycle). After ultrasonication, centrifuge at 12,000 r / min for 20 min at 4°C, take a portion of the supernatant and precipitate, respectively, and perform SDS-PAGE gel electrophoresis to identify whether the protein is in the supernatant or the precipitate.

[0035] (3) Protein purification was performed using the Ni-NTA agarose resin purification kit (Shanghai, China). The specific steps are described in the kit manual. The concentration of the purified protein was estimated by measuring the absorbance at 280 nm. The BSA solution was diluted to a concentration of 1000, 750, 500, 250, 125, 75, 25, and 0 µg / mL. The protein concentration standard curve was prepared using the modified BCA protein concentration determination kit (Shanghai, China). The steps are described in the kit manual. The protein concentration estimated by OD280 was appropriately diluted to a concentration less than 1000 µg / mL. The modified BCA protein concentration determination kit was used to accurately determine the protein concentration after dilution.

[0036] (4) Use 75% alcohol by volume to clean the two glass plates for making the gel. Put the two glass plates together tightly and carefully add 12.5% ​​separation gel from one corner of the glass plate. When the liquid level reaches about 1.5 cm from the top of the glass plate, carefully add deionized water to seal the separation gel and make the surface of the separation gel level. After the separation gel solidifies, use absorbent paper to absorb as much deionized water as possible on the surface of the separation gel, add 5% concentrated gel, and insert a comb.

[0037] (5) After the SDS-PAGE gel solidifies, place it in the electrophoresis tank and add electrophoresis buffer to prepare for sample loading. Use a microinjector to draw 10 µL of sample and add it to the spot well. Run electrophoresis at 90 V for 10 to 20 min. After the indicator moves to the separation gel, increase the voltage to 110 V until the indicator completely runs out of the gel. Continue electrophoresis for 10 min and then stop.

[0038] (6) Strip the SDS-PAGE gel and stain it in Coomassie Brilliant Blue G-250 staining solution for 2 h. Pour away the staining solution, add destaining solution, and destain until the background color of the gel is lighter. Take a photo and observe.

[0039] The results are as follows Figure 3 As shown, the band with a size of 22 kD in the figure is the AmCIP protein.

[0040] The amino acid sequence of the AmCIP protein is as follows: MAGIINKIGETLHVGGNKKEDEHKVESHGDYKGERHDEKTGVLHLGGGKGESHGDYKGENTGVLHGLGGHKGESRGDYKGENTGVLHGLGGHKPDHYGHGEEHKEGFVDKIKDKIHGDPDHVKGEGVVKKKKDKKKHEHGHEHGHDSSSSDSD (SEQ ID No. 1).

[0041] Example 5 The specific steps for the protection and application of AmCIP protein in vitro enzyme activity are as follows: Lactic dehydrogenase (LDH) is an important enzyme in the energy metabolism process in organisms. It catalyzes the interconversion between pyruvate and lactate. The process is accompanied by the interconversion of NAD / NADH. It is widely present in animals and plants. Assay principle: Lactate dehydrogenase can catalyze the dehydrogenation of lactate to produce pyruvate, in which NAD acts as a hydrogen acceptor. Pyruvate reacts with 2,4-dinitrophenylhydrazine to produce pyruvate dinitrophenylhydrazone, which appears brown-red in alkaline solution. The depth of the color is proportional to the concentration of pyruvate, and the activity of lactate dehydrogenase is calculated from this.

[0042] (1) Sample preparation L-Lactate Dehydrogenase (LDH) and Lacate Dehydrogenase Assay Kit were purchased from Solebao to detect the enzyme activity of AmCIP protein-protected LDH. LDH was diluted to 0.68 U / mL using 50 mmol / L sodium phosphate buffer (pH 7.5); AmCIP protein was diluted to a final concentration of 9.3 µmol / L using the Elution Buffer used in the protein purification process; and a 4.63 µmol / L CuCl2 solution was prepared.

[0043] (2) Determination of LDH enzyme activity in the presence of AmCIP protein Take 32 µL of LDH solution (0.68 U / mL) and add 32 µL of AmCIP protein (9.3 µmol / L) solution, mix well; at the same time, use the empty vector purified protein as a negative control, and 50 mmol / L sodium phosphate buffer (pH 7.5) to replace LDH as a blank control. Take 10 µL of enzyme solution as a sample and use the lactate dehydrogenase kit (Solarbio) to measure the enzyme activity. Each protein is repeated in 3 tubes and each tube is repeated 3 times. The experimental steps are shown in the instructions of the lactate dehydrogenase kit. The enzyme activity of the negative control is recorded as 1, and the relative activity of lactate dehydrogenase after adding AmCIP protein is calculated.

[0044] The results are as follows Figure 4 As shown, it indicates that AmCIP protein can significantly enhance the enzymatic activity of LDH.

[0045] (3) AmCIP protein protects LDH enzyme activity under heavy metal copper ion stress Take 32 µL LDH solution (0.68 U / mL), add 16 µL CuCl2 solution (4.63 µmol / L) and mix gently for 30 seconds; add 32 µL AmCIP protein solution (9.3 µmol / L), and use empty vector purified protein as negative control, add 16 µL deionized water instead of CuCl2 solution as positive control, and replace LDH with 50 mmol / L sodium phosphate buffer (pH 7.5) as blank control. Take 10 µL enzyme solution as sample and use lactate dehydrogenase kit (Solarbio) for enzyme activity determination. Each protein is repeated in 3 tubes and each tube is repeated 3 times. The experimental steps are shown in the instructions of lactate dehydrogenase kit. The enzyme activity of the positive control is recorded as 1, and the addition of Cu is calculated. 2+ , the relative activity of lactate dehydrogenase after AmCIP protein.

[0046] The results are as follows Figure 5 (A, B, and C in the figure indicate that there are statistically significant differences among the three treatment groups) 2+ The enzyme activity of LDH was reduced to 45% after the addition of AmCIP protein. The enzyme activity of LDH was protected under the stress of heavy metal copper ions, and the detected enzyme activity was higher than that of the positive control.

[0047] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A cold-induced protein from Ammopiptanthus mongolicus, characterized in that: The amino acid sequence of the cold-induced protein of Ammopiptanthus mongolicus is shown in SEQ ID No.

1.

2. The cold-induced protein of Ammopiptanthus mongolicus according to claim 1, characterized in that: The coding gene sequence of the cold-induced protein of Ammopiptanthus mongolicus is shown in SEQ ID No.

2.

3. The method for preparing the cold-induced protein of Ammopiptanthus mongolicus according to claim 1 or 2, characterized in that: The coding gene shown in SEQ ID No. 2 is introduced into the recipient cell, and the protein is expressed and purified to obtain the product.

4. The method for preparing the cold-induced protein of Ammopiptanthus mongolicus according to claim 3, characterized in that: The Ammopiptanthus mongolicus cold-induced protein is expressed using a prokaryotic expression system.

5. The method for preparing the cold-induced protein of Ammopiptanthus mongolicus according to claim 4, characterized in that: The following steps are involved: (1) Obtaining the target gene: using the cDNA obtained from Ammopiptanthus mongolicus as a template, using primers AmCIP-F and AmCIP-R to perform PCR amplification, and recovering the PCR product to obtain the target gene; the sequences of the primers AmCIP-F and AmCIP-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; (2) Double digestion and ligation: double digestion of the pET28a vector and the target gene obtained in step (1) with restriction endonucleases, and recovery of the double digestion vector and the double digestion target gene; ligation of the double digestion vector and the double digestion target gene with T4 DNA ligase; (3) Obtaining recombinant plasmid: The ligation product obtained in step (2) was transformed into Escherichia coli DH5α for resistance screening, followed by double enzyme digestion and sequencing identification, and the plasmid was extracted to obtain the recombinant plasmid pET28a- AmCIP ; (4) Expression and purification of target protein: The recombinant plasmid pET28a- AmCIP The protein was transformed into Escherichia coli BL21 and expanded, and IPTG was used to induce the expression of the cold-induced protein of Ammopiptanthus mongolicus; the protein was purified using the HIS tag on the protein.

6. Use of the cold-induced protein of Ammopiptanthus mongolicus according to claim 1 or 2 in protecting enzyme activity.

7. The use according to claim 6, characterized in that: The cold-induced protein of Ammopiptanthus mongolicus protects the enzymatic activity of lactate dehydrogenase.

8. The use according to claim 7, characterized in that: The cold-induced protein of Ammopiptanthus mongolicus protects the enzymatic activity of lactate dehydrogenase under heavy metal conditions.

9. The use according to claim 8, characterized in that: The Ammopiptanthus mongolicus cold-induced protein protects the enzymatic activity of lactate dehydrogenase under copper ion conditions.

10. The use according to claim 9, characterized in that: The effective concentration of the cold-induced protein of Ammopiptanthus mongolicus is 3-5 µmol / L.

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